The green algae Chlorella has attracted considerable interest for commercial production of functional foods. Examples include polyunsaturated fatty acids by Chlorella sorokiniana [Chen F and Johns M R, 1991] and lutein by Chlorella protothecoides [Shi X M et al., 2002]. More recently, Chlorella zofingiensis has been proposed as a promising producer for the high-value carotenoid pigment, astaxanthin [Orosa M et al., 2000; Ip, P F et al., 2004]. Astaxanthin (3,3′-dihydroxy-β,β′-carotene-4,4′-dione) (FIG. 1) has been used as feed additives to elicit the pinkish-red color to the flesh of aquatic animals, and to improve their growth and survival in the aquaculture industry [Lorenz R T and Cysewski G R 2000; Pan C H et al., 2001]. Recent studies have also shown that astaxanthin is a potent antioxidant and is effective for the prevention of certain cancers [Tanaka T et al., 1995; Nishino H et al., 1999; Lyons N M and O'Brien N M, 2002]. The annual worldwide market for astaxanthin has been estimated at US$200 million [Lorenz R T and Cysewski G R 2000]. However, astaxanthin is expensive and sells for approximately US$2,500 per kg.
Light has been employed for enhancing astaxanthin formation in algal cultures [Orosa M et al., 2000; Hata N et al., 2001; Zhang D H and Lee Y K, 2001]. However, the attenuated light absorption caused by mutual shading of cells in large-scale cultures severely affects the productivity and quality of algal biomass and products [Chen, 1996; Zaslavskaia L A et al., 2001]. For instance, Harker et al. [1996] reported that the content of astaxanthin in a green alga Haematococcus pluvialis cultivated in a 30-litre photobioreactor was significantly lower than that obtained in a smaller laboratory-scale culture, indicating the insufficiency of illumination in the scaled-up culture system. The high cost of lighting is another problem hindering the commercialization of microalgal products [Borowitzka M A, 1999]. To overcome such a problem, a heterotrophic culture approach may be considered because in heterotrophic culture, light is not needed and the organic substrate serves as the sole carbon and energy source [Chen, 1996; Zaslavskaia L A et al., 2001]. Although heterotrophic cultivation in most Chlorella species has been achieved [Chen F and Johns M R, 1991; Shi X M et al., 2002; Endo H et al., 1977], no literature is available concerning the production of astaxanthin by growing C. zofingiensis in the dark.
A high carbon to nitrogen (C/N) has been suggested to induce astaxanthin biosynthesis because nitrogen limitation in the presence of excess organic carbon substrates such as acetate and glucose has proved effective in enhancing astaxanthin production in mixotrophic cultures [Ip P F et al., 2004; Kakizono T et al., 1992]. While there may be involvement of the additional carbon in the form of CO2 in the photosynthetic process of mixotrophic cultures, CO2 is not involved in the biosynthesis in heterotrophic culture such as the biosynthesis of astaxanthin. Nevertheless, it has now been found that the production of astaxanthin and other carotenoids by Chlorella, especially C. zofingiensis, in dark-heterotrophic culture can be enhanced by increasing the initial C/N ratios in the medium.